BIOESSENTIALS Your guide to vital reagents and equipment for life science applications.

Transcription

1 BIOESSENTIALS Your guide to vital reagents and equipment for life science applications. CELL CULTURE EXTRACTION & PURIFICATION OF BIOMOLECULES PCR & RT-PCR Separation of Nucleic Acids by Agarose Gel Electrophoresis PROTEIN ELECTROPHORESIS

2 Thermo Scientific and Fisher Scientific products deliver an end-to-end solution that can meet your most demanding electrophoresis requirements. You can depend on our expertise in electrophoresis instruments along with ultra-pure reagents that are pre-qualified for your applications. This guide is designed to help you select the right products from our best-in-class array of laboratory equipment and bioreagents. 3-STEP Selection Process Separation of Nucleic Acids by AGAROSE GEL ELECTROPHORESIS 1 Choose Your Reagents Agarose Buffer Ladders 2 Choose Your Equipment Power Supply Gel Box 1 REAGENTS: AGAROSE Choosing your agarose Fisher BioReagents offers three different grades of agarose that are functionally tested and pre-qualified for specific applications. Agarose grades used in electrophoresis of nucleic acids Genetic Analysis Grade agarose that yields biologically active DNA or RNA. Testing includes enzymatic performance measurements. Molecular Biology Grade suitable for analytical separation of DNA or RNA. PCR Grade the original agarose for analytical separation of PCR amplicons (<1kb). Agarose is a linear polysaccharide composed of alternating residues of D- and L-galactose joined by glycosidic linkages. Agarose forms gels that are both porous and resilient. These gel properties provide a sieving matrix which allows the electrophoretic separation of charged macromolecules such as DNA or RNA according to size. Compared to polyacrylamide gel, agarose has a lower resolution but wider range of separation. Lower grades of agarose can be contaminated with other polysaccharides, salts, and proteins. Such impurities can alter the gelling/ melting temperature of agarose solutions or affect the ability to use the recovered nucleic acid sample in a postelectrophoresis application. 3 Downstream Application Essentials Gel Staining Hybridization DNA Gel Extraction 1

3 Two Factors for Selecting an Agarose AGAROSE 1. The size of DNA or RNA fragments to be analyzed (see graph below). Cat. No. BP160 BP165 BP1356 BP1360 BP2410 Agarose Separation Ranges Low EEO/Multipurpose Low Melting/Nucleic Acid Recovery Broad Separation Range for DNA/RNA Low Melting <1kb DNA/RNA Intermediate Melting 500bp to 23kb 200bp to 25kb 500bp to 25kb 50bp to 1kb 15bp to 1.2kb , The type of downstream application that will follow electrophoretic separation (e.g., cloning procedures directly from remelted agarose or in-gel reaction). Agarose Selection Guide Type of Agarose Low EEO Low Melting Low Melting Wide PCR >200bp <1000bp Separation Grade Range Cat. No. BP160 BP165 BP1360 BP1356 BP2410 Recovery of DNA and RNA x x x x x Southern and Northern Blots x DNA/RNA separation 50bp to 1kb x x DNA/RNA separation >1kb x x x PCR fragment analysis x x x x x In-gel reactions (ligation, transformations, PCR) x Colony lifts x Available pack sizes 100g and 500g 25g 100g 100g and 500g 100g Agarose grade Molecular Molecular Genetic Genetic PCR biology biology analysis analysis 2

4 1 REAGENTS: BUFFERS Choosing your buffers Our Fisher BioReagents line of electrophoresis buffers is available in various package configurations to suit all budgets. Choose from the most economical powder components, through concentrated stock solutions, to ready-to-use concentrations in specially designed containers featuring faucets for easy dispensing. Two buffers commonly used for DNA agarose electrophoresis are Tris-acetate with EDTA (TAE; 40mM Tris-acetate, 1mM EDTA) and Tris-borate with EDTA (TBE, 89mM Tris-borate, 2mM EDTA). Because the ph of these buffers is neutral, the phosphate backbone of DNA has a net negative charge and migrates toward the anode. TAE and TBE have different properties which makes one more suitable than the other for a specific purpose. MOPS is a commonly-used buffer system for RNA electrophoresis using formaldehyde or formamide denatured RNA. It is important to use RNase-free chemicals, water, and containers when preparing the buffer solution. The typical formulation of a 10X MOPS running buffer is 0.4M MOPS (ph 7.0), 0.1M sodium acetate, and 0.01M EDTA. The denaturing system chosen depends on the purpose of the RNA experiment and the size of the RNA fragment being separated. Formaldehyde denaturation is suitable if RNA samples are to be recovered. Formamide denaturation is suitable if the RNA needs to retain its biological activity. Buffer Suggest Uses Properties TAE DNA recovery. Low buffering capacity. Electrophoresis of large DNA (>12 kb). Recirculation may be necessary for extended run times (>6 hr). TBE Electrophoresis of small DNA (< 1kb). Decreased DNA mobility. Increased resolution of small DNA (< 1kb). High buffering capacity no recirculation required for extended run times. MOPS Electrophoresis of formaldehyde denatured RNA. Buffer is low in ionic strength. Recirculation of buffer may be necessary. 3

5 Buffers for Nucleic Acid Applications BUFFERS Cat. No. Concentration Tris- Borate- EDTA TBE BP X 1L BP X 4L BP X 20L BP X 1L* BP X 1L BP X 4L BP X 20L BP X 1L** Cat. No. Concentration Tris- Borate- EDTA TAE BP X 4L BP X 20L BP X 500mL BP X 1L BP X 4L BP X 20L BP X 1L BP X 500mL BP X 1L BP X 4L BP X 20L BP X 1L** Cat. No. Description Size MOPS BP Powder 100g BP Powder 500g Water BP Nuclease-Free 50mL BP Nuclease-Free 100mL BP DNA-Grade 1L BP561-1 RNA-Grade 1L Formaldehyde BP % by weight 25mL BP % by weight 500mL *Pre-weighed powder in poly bottle. Dissolve in water. ** Pre-weighed powder in foil pack. Dissolve in water. Suggested Agarose Concentrations The optimal gel concentration depends on the size of the DNA fragments to be resolved. Cat. No. Main Application DNA Size Range Finale Agarose Finale Agarose in Base Pairs Concentration % (W/V) Concentration % (W/V) 1x TAE buffer 1x TBE buffer BP1360 Low melting temperature agarose , Certified recovery of small nucleic acid fragments Outstanding resolution BP165 Low melting temperature agarose , Broad separation range , Ideal for DNA and RNA recovery , after electrophoretic separation , , , BP1356 Suitable for routine nucleic acid 1,000-23, BP160 electrophoresis applications with , broad separation range , , , ,

6 1 REAGENTS: LADDERS Choosing your ladders To achieve the most accurate qualitative and quantitative analysis via agarose gel electrophoresis, the appropriate DNA or RNA standard is required. Fisher BioReagents provides a wide range of standards, including routine DNA ladders for quick size and quality assessment as well as exactgene DNA ladders that allow for quantitative analysis. exactgene and Routine DNA Ladders Ready-to-use (pre-mixed with the loading dye), room temperature, stable DNA ladders are available for all common electrophoresis applications. Cat. No. Application Size Number Number Range of Bands of Loadings exactgene DNA ladders are ideal for qualitative analysis, quantitative estimation, and size assessment BP PCR fragment analysis bp /10uL BP PCR fragment analysis, small DNA digests bp /10uL BP Quick check of PCR or enzyme digestion results bp 8 100/10uL BP General purpose, small DNA fragments bp /10uL BP Fast run times, small DNA fragments bp /10uL BP Clone identification bp /10uL BP Large size PCR or cloning bp /10uL BP Small and large cloning application bp /10uL BP General purpose, large digested DNA ,000bp /10uL BP General purpose, wide size range ,000bp /10uL BP General purpose, extra-large fragments ,000bp /10uL Routine DNA ladders are designed for qualitative analysis and size assessment BP Small fragments, quick size assessment bp /5uL BP Quick size assessment of broad size range 50-10,000bp /5uL For Lambda DNA digests or other DNA markers and ladders not containing loading dye, please visit and type: BP in the search box. 5

7 LADDERS RiboLadders RNA Standards These standards can be used to assess single-stranded RNA molecules on both native and denaturing agarose gels. These unique RNA standards are lyophilized to reduce thawing-related degradation, to prolong shelf life and to ensure consistent performance. Cat. No. Application Size Number Number Range of Bands of Loadings Sizing unknown RNA fragments BP Small RNA fragments 0.1 1kb 8 50 BP Large RNA fragments 0.2 4kb 9 50 Routine DNA Ladders exactgene DNA Ladders BP2579 RiboLadders RNA Standards BP2581 BP2582 BP2810 BP2811 Bases Bases Bases Bases 2,000 10,000 1,000 1,550 8,000 4,000 1,400 6,000 3, ,000 1,000 2,000 3, , bp 10, , , , , , , , , , ng/10µl 2,000 1,550 1,400 1, ,000 BP2578 bp ng/10µl BP2573 bp ng/10µl BP2571 bp 10, , , , , , , , , , , , ng/10µl 6

8 2 EQUIPMENT: GEL BOXES AND POWER SUPPLIES Choosing your gel box and power supply Submarine Electrophoresis Gel Boxes Thermo Scientific submarine electrophoresis gel boxes are available in three sizes and sample quantities. This gel size flexibility is important in order to provide sufficient separation of closely-spaced DNA fragments as well as small and large nucleic acid fragments on the same gel. Cat. No. Model Cooling Max Number Gel Size Running Buffer Option of Samples (W x L) in cm Volume in ml EasyCast B1 no 34 9 x OWB1BP EasyCast B1 yes 34 9 x B EasyCast B2 no 108 with rapid load tray 12 x EasyCast B2 yes 108 with rapid load tray 12 x EasyCast B3 yes 300 with rapid load tray 12 x OWA6 Millipede A6 no x EasyCast Systems: Compact footprint EasyCast gasket UVT gel tray SuperSafe lid with attached power supply leads Optional cooling system Millipede System: Buffer chamber with 3-point leveling base SuperSafe lid with attached power supply leads UV transparent gel tray with gasket end gates Leveling bubble Voltage Table The table below provides recommended voltages and buffers according to DNA size and application. The distance used to determine the voltage gradients is the distance between electrodes, not the gel length. If the voltage is too high, band streaking may occur for large DNA sizes (>12kb). When the voltage is too low, the mobility of small (< 1kb) DNA is reduced, and band broadening will occur due to dispersion and diffusion. Gel Size Voltage Recovery Analytical Buffer Buffer <1kb 5 V/cm TAE TBE <1 kb to >12 kb 4-10 V/cm TAE TBE >12 kb 1-2 V/cm TAE TAE Easycast B1 Gel Box 7

9 Low Voltage Power Supplies 105 Power Supply Suitable for DNA/RNA submarine gel electrophoresis and mini-sds PAGE Power Supply Designed to operate in constant voltage or constant current with a continuously variable output in either mode. Appropriate for DNA/RNA submarine gels, electroblotting mini gels, and mini protein and mini SDS PAGE. 105 and Power Supplies Each unit is capable of automatically crossing over from one mode of operation to the other when the preset limit is reached. EQUIPMENT Power Supply Capable of producing up to 500 volts and 500 milliamps, this power supply is ideal for PAGE, SDS-PAGE, DNA or RNA submarine gels, as well as all types of small-format transfer apparatus Power Supply This unit is capable of producing up to 500 volts or 2,500 milliamps and is an excellent choice for running all types of electroblotting applications. It is also ideal for PAGE, SDS-PAGE, and DNA or RNA submarine gels and Power Supplies Both models prevent sample diffusion with a unique holding voltage feature. Samples and systems are protected by an automatic crossover function that automatically switches over from one mode of operation to the other when the preset limit is reached. Enables efficient time management with dual-function, 100-hour timer. Simple operation a single knob controls the output, and a single pushbutton selects the mode to be adjusted. 105 Power Supply Power Supply Power Supply Specifications Operating Voltage Low range (10 V steps) Volts 500 Volts 500 Volts High range (10 V steps) Maximum Voltage 250 Volts Operating Current Range 500 ma 2500 ma Maximum Output Power 125 Watts 250 watts 325 watts Maximum Current V milliamps 500 milliamps below 150 V No. of Output 2 sets of connectors 4 sets of recessed 4 sets of recessed 4 sets of recessed Terminals (female) 4 mm connectors 4 mm connectors 4 mm connectors 4 mm connectors Mode of Operation Constant voltage or constant current with automatic crossover Safety Interlock Load sensing shut down Load sensing shut down disconnect. Automatic disconnect. Automatic restart provided in case of power failure. restart provided in case Key actuation necessary to activate high voltage. of power failure. Timer Dual function up/down timer which deactivates high voltage upon reaching zero time remaining in count down mode hour. 99 hr 59 min 99 hr 59 min Dimensions (w x d x h) 213 x 133 x 114 mm 220 x 190 x 140 mm 225 x 303 x 145 mm 225 x 303 x 145 mm Weight 2.3 kg net, 3.2 kg shipping 2.3 kg net, 6 kg shipping 8.4 kg 8.4 kg Voltage Cat. No. Cat. No. Cat. No. Cat. No. 115 V 105ECA ECA ECA ECA V 105ECA-LVD 25090ECA-LVD 13590ECA-LVD 57090ECA-LVD 8

10 3 DOWNSTREAM APPLICATION ESSENTIALS Choosing your application Post-Electrophoresis Applications and Related Supplies There are multiple applications that follow nucleic acid electrophoresis. Thermo Scientific offers a broad selection of equipment along with essential Fisher BioReagents for these protocols. Gel Staining This process can be done during electrophoresis by adding stains such as ethidium bromide into the gel. Alternatively the gel is stained after the separation is completed. Fisher BioReagents offers ethidium bromide in a solution that saves time and limits manipulation of the carcinogenic chemical. To accelerate the staining and destaining of gel, the gel tray can be shaken using a multi-purpose shaker. Hybridization DNA or RNA is transferred from the agarose gel to a membrane for Southern or Northern hybridization. The hybridization process is carried out in a temperaturecontrolled chamber such as the Thermo Scientific Hybaid Shake & Stack. This oven provides unmatched reliability, accuracy and uniformity to ensure reproducible results. Fisher BioReagents offers functionally tested reagents and buffers for the hybridization process. DNA Gel Extraction In certain cases there is a need to extract the specific size DNA fragment from the gel. The piece of agarose that contains the DNA is melted at a higher temperature in the presence of high salt buffer and then binds to the spin column. The liquid is passed through the column by centrifugation, which is the fastest and most efficient method to extract the DNA. Our Thermo Scientific Sorvall Legend Micro 17 & 21 microcentrifuge series provides fast and efficient processing of your micro-volume samples. 9

11 Thermo Scientific and Fisher Scientific offer a complete LABORATORY SOLUTION Thermo Scientific Millipede Horizontal Electrophoresis System Thermo Scientific Hybaid Shake & Stack Fisher BioReagents Buffers Thermo Scientific Sorvall Legend Micro 17 & 21 Microcentrifuge Series For more information on Fisher BioReagents go to For Thermo Scientific equipment visit 10

12 SPECIAL PROMOTION FREE Fisher BioReagents Buy any electrophoresis equipment listed below and receive FREE Reagents worth up to 50% of your purchase price. act now! 1 Purchase any of the electrophoresis instruments listed below through Fisher Scientific by 9/30/ Visit and choose your FREE Fisher BioReagents from a list of over 500 products. FREE Fisher BioReagents Product Cat. No. Value Thermo Scientific Power Supplies Model 105 FB105 $300 Model FBEC13590 $500 Model FBEC25090 $300 Model FBEC57090 $500 Model 3000P FBEC3000P $600 Thermo Scientific Gel Boxes Easy Cast B $150 Easy Cast B1 with cooling option OWB1BP $160 Easy Cast B B $200 Easy Cast B2 with cooling option $220 Easy Cast B $225 Millipede A6 OWA6 $250 Related Thermo Scientific Equipment Sorvall Legend Micro $300 Sorvall Legend Micro $300 Sorvall Legend Micro 17R $400 Sorvall Legend Micro 21R $400 Legend 14 Microcentrifuge $400 Hybaid Shake & Stack HBSNSR $500 Hybaid Shake & Stack (+platform) HBSNSRS $550 Reagents Agarose - 500g size BP $150 BP $150 BP $150 DNA or RNA Ladder (listed in this brochure) $50 Offer expires on 09/30/2007. Free BioReagents must be redeemed by 10/31/2007. Valid in the U.S. only. Not valid with any other offers, contracts or agreements. Void where prohibited. Free Fisher BioReagents value based on US listed prices Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. BN BRO-SPSBIOGE-0307